Efficacy of Wex-cide 128 disinfectant against multiple prion strains

Prion diseases are transmissible, fatal neurologic diseases that include Creutzfeldt-Jakob Disease (CJD) in humans, chronic wasting disease (CWD) in cervids, bovine spongiform encephalopathy (BSE) in cattle and scrapie in sheep. Prions are extremely difficult to inactivate and established methods to reduce prion infectivity are often dangerous, caustic, expensive, or impractical. Identifying viable and safe methods for treating prion contaminated materials is important for hospitals, research facilities, biologists, hunters, and meat-processors. For three decades, some prion researchers have used a phenolic product called Environ LpH (eLpH) to inactivate prions. ELpH has been discontinued, but a similar product, Wex-cide 128, containing the similar phenolic chemicals as eLpH is now available. In the current study, we directly compared the anti-prion efficacy of eLpH and Wex-cide 128 against prions from four different species (hamster 263K, cervid CWD, mouse 22L and human CJD). Decontamination was performed on either prion infected brain homogenates or prion contaminated steel wires and mouse bioassay was used to quantify the remaining prion infectivity. Our data show that both eLpH and Wex-cide 128 removed 4.0–5.5 logs of prion infectivity from 22L, CWD and 263K prion homogenates, but only about 1.25–1.50 logs of prion infectivity from human sporadic CJD. Wex-cide 128 is a viable substitute for inactivation of most prions from most species, but the resistance of CJD to phenolic inactivation is a concern and emphasizes the fact that inactivation methods should be confirmed for each target prion strain.


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Unfunded studies Enter: The author(s) received no specific funding for this work. Experimentation followed RML IACUC approved protocol #2021-003-E. Mice were euthanized by inhalation isoflurane overdose followed by cervical dislocation. ELpH has been discontinued, but a similar product, Wex-cide 128, containing the similar 29 phenolic chemicals as eLpH is now available. In the current study, we directly compared the 30 anti-prion efficacy of eLpH and Wex-cide 128 against prions from four different species 31 (hamster 263K, cervid CWD, mouse 22L and human CJD). Decontamination was performed on 32 either prion infected brain homogenates or prion contaminated steel wires and mouse bioassay 33 was used to quantify the remaining prion infectivity. Our data show that both eLpH and Wex-34 cide 128 removed 4.0-5.5 logs of prion infectivity from 22L, CWD and 263K prion 35 homogenates, but only about 1.25-1.50 logs of prion infectivity from human sporadic CJD.

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Wex-cide 128 is a viable substitute for inactivation of most prions from most species, but the 37 resistance of CJD to phenolic inactivation is a concern and emphasizes the fact that inactivation 38 methods should be confirmed for each target prion strain.

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Prion diseases, also known as transmissible spongiform encephalopathies (TSE), are 44 unique infectious diseases that occur following the repeated conversion of normal host derived 45 cellular prion protein (PrPC) into a mis-folded, protease-resistant, infectious, disease associated 46 conformation (PrPSc) [1]. Unfortunately, infectious prions are inherently difficult to inactivate  Environ LpH is much less caustic to equipment and not as toxic or cumbersome to handle 57 and discard appropriately. Importantly, eLpH has the ability to remove greater than 10 7 LD50s of 58 prion infectivity from 263K scrapie infected hamster brain homogenate [2]. The mode of action 59 for eLpH against prion inactivation was never identified, but research on several other phenolic 60 products in the LpH series showed poor anti-prion activity [4]. Unfortunately, production of 61 eLpH has been discontinued. However, a similar phenolic product, which includes two of the 62 same phenols (Ortho-benzyl-para-chlorophenol (BP) and O-phenylphenol (OPP)) present in 63 eLpH is now available from Wexford Labs marketed as Wex-cide 128. Wex-cide 128 is an EPA 64 4 registered pesticide marketed as a disinfectant, deodorizer and cleaner for healthcare, schools, 65 and industry. We were interested in the potential of Wex-cide 128 as a prion disinfectant.

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In the current study, we compared the efficacy of Wex-cide 128 to eLpH against 67 infectious prions derived from four different species: hamster adapted 263K scrapie, white-tailed 68 deer Chronic Wasting Disease (CWD), mouse-adapted scrapie strain 22L, and one human prion 69 strain, MM1 sporadic Creutzfeldt-Jacob Disease (sCJD). We tested all four prion strains by 70 decontaminating prion infected brain homogenates followed by animal bioassay to measure 71 remaining prion infectivity. For two of the strains, 263K and sCJD, we also tested inactivation 72 of prions bound to steel wires. Steel wires act as a surrogate for surgical instruments and have a 73 non-porous surface similar to many coatings present in laboratories and hospitals. Our results 74 using mouse bioassays showed that both eLpH and Wex-cide 128 were highly efficacious and 75 suitable for inactivation of CWD, 22L and 263K prions, but much less effective against human 76 sCJD. The discovery that sCJD was more resistant to inactivation by phenolic disinfectants is a 77 concern and reaffirms that prion disinfectant efficacy must be verified for each target prion 78 strain/species, as not all infectious prions are inactivated equally [5-10].

Efficacy and shelf life of Wex-cide 128 against 263K hamster prions 82
Wex-cide 128 is typically used at a 1:128 dilution (~0.8%) for general disinfectant 83 applications. However, in our studies we tested a 4% dilution of Wex-cide 128 in order to 84 normalize the BP concentration to what is present in 2% eLpH (Table 1). Environ LpH has 85 previously established efficacy against 263K hamster prions and is routinely used in our 86 5 laboratory as a 2% solution to inactivate prions. We have included 2% eLpH in the current study 87 as a prion inactivation control and experimental group for historical/experimental comparison. 88 We also tested Wex-cide 128 at a ten-fold higher concentration (40%) to better understand the 89 level of phenols necessary for anti-prion activity. Ten percent 263K-infected brain homogenates 90 were mixed at a 1:9 ratio of brain homogenate to disinfectant for 30 minutes. After this 91 decontamination step, the brain homogenate/disinfectant mixture was further diluted and 92 immediately inoculated intracerebrally into tg7 mice. Additional dilution was necessary to 93 prevent acute toxicity in recipient bioassay mice due to the residual disinfectant. As a no 94 treatment control, 263K brain homogenate was treated with saline for 30 minutes prior to 95 dilution and inoculation. Our data showed 4% Wex-cide 128 and 2% eLpH both reduced 263K infectivity by over 105 5 logs compared to saline treatment alone (Table 2.). Importantly, our data also showed no 106 added benefit to using 40% Wex-cide over 4% against 263K prions. Interestingly, one mouse 107 inoculated with a 10 -3 dilution of eLpH treated 263K did develop prion disease at a late time 108 (Table 2). To our knowledge, this is the first time eLpH failed to inactivate prions in brain 109 homogenate to below detectable limits, [2, 4, 11] but the stock eLpH used for these studies was  Table 2. Bioassay of disinfected 263K brain homogenate in tg7 mice 119 120 a Aliquots of 263K brain homogenates (10%) were exposed to different disinfectants or saline for 30 121 minutes at a 1:9 ratio. Solutions were then further diluted for bioassay in mice. Each recipient mouse 122 received 30µl of inoculum. 123 b The calculated titer reported is the log10LD50 / gram of tissue 124 c The numerator is the number of prion-positive mice (see methods), and the denominator is the 125 number of mice inoculated. For groups with positive mice the average incubation period in days-post 126 inoculation (dpi) is provided.  Table 5. Bioassay of disinfected 22L brain homogenate in tga20 mice 213 214 a Aliquots of 22L-infected brain homogenates (10%) were exposed to different disinfectants or saline for 215 30 minutes at a 1:9 ratio. Solutions were then further diluted for bioassay in mice. Each recipient 216 mouse received 30µl of inoculum. 217 b The calculated titer reported is the log10LD50 / gram of tissue 218 c The numerator is the number of prion-positive mice (see methods), and the denominator is the 219 number of mice inoculated. For groups with positive mice the average incubation period in days-post 220 inoculation ( Efficacy of Wex-cide 128 against sCJD brain homogenates and prion coated steel wires 225 Decontamination of human tropic prions is an important biomedical and research 226 biosafety concern. We therefore tested the ability of Wex-cide 128 and eLpH to inactivate sCJD 227 prions derived from transgenic mice that expressed human prion protein with methionine at 228 codon 129. We tested inactivation of sCJD in brain homogenates and also bound to stainless 229 steel wires. We found that sCJD brain homogenates treated for 30 minutes with 2% eLpH or 4%

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Wex-cide 128 showed only a slight reduction in prion infectivity of 1.25 logs (Table 6). Using a 231 ten-fold higher concentration of Wex-cide 128 only improved the reduction in sCJD prion 232 infectivity by an additional 0.25 logs. Inactivation of steel wire bound sCJD prions was more 233 effective. We achieved complete removal of prion infectivity when the sCJD coated wires were 234 immersed for 30 minutes in either eLpH or Wex-cide 128 (Table 7). Two minutes in these same 235 disinfectants was not as effective, as several mice with treated, implanted wires developed sCJD 236 (Table 7). In contrast to the 263K coated wires shown in table 3, wires coated with 10-fold serial 237 dilutions of sCJD did not correlate with increasing incubation periods as the wires were exposed 238 to less sCJD ( Table 7). Because of this non-linear response, and failure to reach an end-point in 239 our no treatment control we did not attempt to extrapolate a decrease in titer for this experiment.   a Steel wires were exposed to sCJD prion infected brain homogenates, then washed, dried, and 272 immersed in different disinfectants for either 2 or 30 minutes. Following treatment wires were removed 273 and allowed to dry. Each mouse was implanted intracerebrally with a single 3-4 mm wire. Dilution of sCJD brain homogenate used to coat wires, prior to treatment a 10 -1 10 -3 10 -4 10 -5 was a clear candidate to screen as it contained the same two phenols that are the main 287 components in eLpH (Table 1).

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In our experiments, a 30-minute treatment using 4% Wex-cide 128 reduced prion 289 infectivity from brain homogenates by at least 10 4 infectious units for 263K, 22L and CWD 290 ( Table 8). Many of these reductions are likely to be underestimations as our lower limit of 291 detection in the assay is dictated by acute toxicity from the disinfectant and prevents us from 292 testing more concentrated samples. Wex-cide 128 treatment also removed at least 10 6 infectious 293 units from 263K coated steel wires (Table 3). Compared to eLpH, Wex-cide 128 was equal to or 294 superior to eLpH in reduction of infectious prions derived from four different species (Table 8).

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Wex-cide 128 also demonstrated stable shelf life of at least 8 months following dilution to a 4% 296 working concentration ( The utility of wires as a surrogate for surfaces and instruments have been used with 311 success by several other groups testing prion inactivation [5,8,[13][14][15][16][17]. Our studies using 263K 312 coated steel wires had a clear advantage in sensitivity over the homogenate bioassays (Tables   313   2&3). Following prion coating and subsequent decontamination, the steel wires can be dried, 314 effectively eliminating residual decontaminate solution. This feature allows higher sensitivity, as 315 decontaminated brain homogenates must be diluted prior to inoculation in mice to avoid acute 316 toxicity. In the current study we found that 263K prions and sCJD prions appear to have 317 differing affinities for binding steel. Bioassay data from implanted wires coated with 10-fold 318 decreasing concentrations of 263K dilutions showed that wires exposed to fewer 263K prions 319 bound less infectious 263K prions based on incubation periods in mice (Table 3). This data was 320 also similar to previous studies using 263K or vCJD coated wires [9,11]. The same trend was 321 not observed in our wire experiments with sCJD, where decreasing concentrations of sCJD did 322 not correspond to less sCJD infectivity on the wire. We are not certain why this is, but postulate 323 that sCJD prions bind the wire with higher affinity, as different prion strains have been 324 documented to bind metals differentially [18]. Unfortunately, we did not reach an endpoint in 325 our control wire bioassay (Table 7). But even if an endpoint had been reached, the non-linear 326 survival times with the sCJD control wires made estimation of reductions in titers unreliable for 327 this experiment.

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While identification of the key chemical responsible for inactivation of prions was not a 329 primary goal of our project, we believe that data from our current study combined with previous 330 work identifies the likely anti-prion phenol. Only two phenol derivatives are present in Wexcide 331 128, BP at 3.03% and OPP at 3.4%. Previous work showed that a phenolic mixture that 332 contained OPP at 7.7%, but no BP, had no reduction in prion infectivity [4]. By deduction, the 333 20 phenolic component of Wex-cide 128 that provides the most anti-prion properties is likely BP.

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Environmental Protection Agency (EPA) mandated a full phase-out of PTAP in EPA registered 336 pesticides.

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The discovery that neither eLpH or Wex-cide 128 were very effective against sCJD brain 338 homogenates was a concern, but not entirely surprising. Previous studies have shown that 339 different prion strains can differ in resistance to inactivation [5][6][7][8][9][10]19]. Of particular interest 340 were the data showing that sCJD was 10,000-100,000 times more resistant to acidic SDS 341 inactivation compared to hamster scrapie [5,8]. The ability of sCJD to resist acidic SDS 342 treatment and phenolic chemicals demonstrated that sCJD may also be more difficult to 343 inactivate using other currently approved methods.

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As a biosafety precaution we reviewed the literature specific to chemical inactivation of 345 sCJD. Several manuscripts from years ago reported concentrated bleach decreased CJD prion 346 infectivity by 3-4 logs [20][21][22]. Unfortunately, the CJD tested in these studies was not directly 347 derived from human brain, but was instead obtained from CJD that had been adapted to either 348 guinea pigs [20,21] or mice [20,22]. Guinea pigs and mice both have very different PrPC 349 amino acid sequences compared to humans. This was of great concern as we now understand 350 that passaging a prion strain into a novel host does not guarantee the prion strain, protein folding, 351 or susceptibility to inactivation will remain consistent with the original strain. were anesthetized with isoflurane and inoculated in the left-brain hemisphere with 30 μl of 407 disinfectant-treated or saline-treated brain homogenate dilutions.

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Decontamination and bioassay of 263K or sCJD coated steel wires 410 Sterile stainless steel suture wires (Havel, size 000), cut into 3-4 mm lengths, were 411 immersed in either 263K or sCJD 10% brain homogenates for one hour with gentle agitation.

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Following immersion, the prion infected brain homogenates were removed using a pipette and 413 the wires were washed briefly in an excess of sterile water. The water was drawn off and the 414 wires were allowed to air dry in a sterile petri dish. To decontaminate the wires, wires were 415 submerged in disinfectants (4% Wex-cide 128 or 2% eLpH) for either 2 or 30 minutes. Saline 416 was used as a mock disinfectant. To create standard curves for the levels of prion infectivity able 417 to bind to the steel wires, wires were exposed to ten-fold dilutions of 263K brain homogenate 418 (10 −1-10 −7 ) or sCJD brain homogenate (10 -1 -10 -5 ). Wires for each experimental group were put 419 into 3-8 recipient mice as shown in tables 3&7. 263K treated wires were implanted into tg7 420 mice while sCJD treated wires were implanted into tg66 mice. Wire implantation and pain 421 management was performed as previously described [11].  Table 1). Screened mice that showed clinical signs of 440 prion disease and had evidence of prion infection were scored as positive in the tables 2-7.

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Different screening methods were used for the four different mouse models (below paragraphs).

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Mice that did not have clinical signs consistent with end-stage prion disease but did have 443 evidence for prion disease based on a prion screening test were not included as positive mice in 444 the bioassay tables. This subclinical situation was very rare, and only occurred with three 445 individual mice that were part of shelf-life experiments (Supplementary Table 1).